Pharmacokinetics of the Recalcitrant Drug Lamotrigine: Identification and Distribution of Metabolites in Cucumber Plants

Treated wastewater is an important source of water for irrigation. As a result, irrigated crops are chronically exposed to wastewater-derived pharmaceuticals, such as the anticonvulsant drug lamotrigine. Lamotrigine is known to be taken up by plants, but its plant-derived metabolites and their distribution in different plant organs are unknown. This study aimed to detect and identify metabolites of lamotrigine in cucumber plants grown for 35 days in a hydroponic solution by using LC-MS/MS (Orbitrap) analysis. Our data showed that 96% of the lamotrigine taken up was metabolized. Sixteen metabolites possessing a lamotrigine core structure were detected. Reference standards confirmed two; five were tentatively identified, and nine molecular formulas were assigned. The data suggest that lamotrigine is metabolized via N-carbamylation, N-glucosidation, N-alkylation, N-formylation, N-oxidation, and amidine hydrolysis. The metabolites LTG-N2-oxide, M284, M312, and M370 were most likely produced in the roots and were translocated to the leaves. Metabolites M272, M312, M314, M354, M368, M370, and M418 were dominant in leaves. Only a few metabolites were detected in the fruits. With an increasing exposure time, lamotrigine leaf concentrations decreased because of continuous metabolism. Our data showed that the metabolism of lamotrigine in a plant is fast and that a majority of metabolites are concentrated in the roots and leaves.


Table S2. Multiple reaction monitoring (MRM
Scheme S3: The reaction scheme and conditions for the synthesis of the M284.
The retention time and MS/MS spectra did not match Acetone The retention time and MS/MS spectra did not match ACN ACN Scheme S4: The reaction scheme and conditions for the synthesis of the M312.

M314
Scheme S5: The reaction scheme and conditions for the synthesis of the M314.Scheme S6: The reaction scheme and conditions for the synthesis of the M418.

IDENTIFICATION OF LTG METABOLITES
Fragmentation patterns of lamotrigine metabolites after expose of cucumber plants during 18 days, are presented.In the first section the MS/MS spectrum of identified metabolites are presented in comparison to a synthesized or a commercial standard (Figures S1-S7).In the following, the mass spectral information of the detected metabolites is presented (Figures S8- S16).All fragment ions colored in blue were observed in the fragmentation pattern of lamotrigine or lamotrigine metabolites. 1 The fragment ion at m/z 256.01 represents the mass of lamotrigine in its ionic state.

LTG-N2-Methyl
Mass spectrum of the M270 exhibited a quasi-molecular ion at m/z 270.0308 [M+H] + in positive-ion mode and the molecular formula was identified as C10H10N5Cl2 (LTG+CH2) by high resolution MS (Table 1).The transformation probably suited to N-methylation of LTG.
LTG-N2-methyl was found as a minor metabolite in human urine as reported by Zonja et al. 2 The fragmentation patterns of the metabolite observed in the plants extract were compared to the fragmentation pattern of the commercially available standard, LTG-N2-methyl.The comparison between the fragmentation patterns (Figure S1D vs. Figure S1E) and retention times (Figure S1A vs. Figure S1B) were executed and revealed a good match between the commercially standard and the M270, which was observed in the plant extract.Therefore, the confidence level is 1.  1).This modification could be suited to Nmethylation of OXO-LTG.M271 was reported by Zonja et al. 2 in wastewater.As a commercial standard was not exist, we synthesized OXO-LTG-Me using OXO-LTG starting material and methyl iodide.The reaction scheme and conditions are provided in Scheme S2.The mass spectral data of M271 observed in the plants was compared to the synthesized standard.Both the retention time and the mass spectral fingerprint were matched (Figure S2).Full-MS chromatogram along with the ESI-MS/MS spectrum of M271 at a collision energy of 30 eV are depicted in Figure S2A and Figure S2D, respectively.The suggested structure (Figure S2C), Full-MS chromatogram along with the ESI-MS/MS spectrum of the synthesized standard spiked into the control sample at a collision energy of 30 eV are depicted in Figure S2B and  LTG-N2-oxide Four chromatography peaks, eluted at different retention times with high-resolution mass spectrometry (HRMS) of m/z 272.0099, were identified as the oxidation products of LTG (LTG+O), as detailed in Table 1 and Figure S3A.The fragmentation patterns of the four isomers observed in the plants extract were compared to the fragmentation pattern of the commercially available standard, LTG-N2-oxide.The ESI-MS/MS of peak observed at retention time 14.6 min (Figure S3D) and the ESI-MS/MS of the commercially available standard (Figure S3E) were completely matched.In addition, spiking of the commercially available standard LTG-N2-oxide into the control samples was carried out revealing a chromatographic peak at the retention time of 14.6 min (Figure S3B).As the result, the structure of the metabolite was confirmed and the confidence level is 1. corresponded to the loss of carbonyl (CO) from the precursor ion (data not shown).At a higher collision energy (e.g., 40 eV), in addition to ion at m/z 256.0149, other product ions representative of the LTG skeleton were also observed (Figure S4D).Thus, the CO moiety is suggested to conjugate to the N2-LTG.Loss of carbonyl as a result of a cleavage at the C-N bond of the formamide group to generate the intact LTG is a common dissociation process 3 and a predicted structure was proposed (Figure S4C).To verify our assumption, the synthesis of LTG-formamide conjugate was produced under reflux of LTG with the formamide building block at basic conditions (see Scheme S3).As aforementioned for other metabolites we compared the fragmentation patterns of the peak observed in the plants extract (Figure S4D) to the fragmentation pattern of the synthesized standard spiked into the control sample (extract root of cucumber plants without exposure to LTG, Figure S4E).Ought to the identical MS/MS spectra match along with an identical retention time (r.t 16.8 min, Figure S4A versus Figure S4C) we deduced that our proposed structure is correct and the structure of M284 has been confirmed with confidence level 3.As aforementioned for other LTG-conjugates, the position of the carbonyl substituent is not determined.positive-ion mode and the molecular formula was identified as C12H12ON5Cl2 (LTG+ C3H4O, Table 1).The ESI-MS/MS spectra exhibited the formation of three product ions with higher masses than the product ion at m/z 256.0149 which was attributed to the intact protonated LTG.

S12
These three ions along with ion at m/z 256.0149 were the key role in structural elucidation process.The first was an ion at m/z 294.0305 which corresponded to loss of water from the m/z 312.0411 precursor ion; the second was an ion at m/z 268.0158 which corresponded to loss of C2H4O (epoxide or acetaldehyde) from the precursor ion; and the third ion was at m/z 266.0125.
Loss of C3H4O from m/z 312.0411 precursor ion to produce product ion at m/z 256.0149, may be suited to the loss of acrylaldehyde as a result of a cleavage at the C-N bond of a potential amide group.As a result of the MS/MS data interpretation, three possible structures were raised as options.The first predicted structure of M312 was the formation of a propionamide moiety (a conjugation of propan-1-one group to one of the three amine nitrogen groups of LTG (Scheme S4A).Propionyl chloride was utilized as a reagent to react with LTG to produce the LTG-N2-propan-1-one.The synthetic route is provided in the supporting information (Scheme S4A).The retention time and the MS/MS spectra of M312 in the plant's extract and the synthesized standard did not matched.Consequently, we predicted an alternative structure.The alternative candidate structure might have contained two groups: propenyl group attached to on nitrogen at the N2-position and a hydroxyl group bound to the primary amine to produce a hydroxyl amine moiety (Scheme S4B).These two-step reactions involved the N-alkylation of LTG with allyl bromide followed by oxidation of the primary amine to form a hydroxyl amine.
The proposed structure and the synthetic route are depicted in Scheme S4B.However, fragment ions ratio and retention times were not identical.The third proposed structure was the attachment of propene-1-ol to the LTG moiety which may tautomatizes (tautomer equilibrium) to form N-propionaldehyde moiety (a conjugation of propanal to one of the three amine nitrogen groups of LTG (Scheme S4C).Notably, the plausible structures of product ions at m/z 294.0303 and at m/z 268.0148 that were observed in the ESI-MS/MS of M312 were also detected in the ESI spectra of lamotrigine-N2-glucurunide TP430 by Zonja et al., 2 and that strengthened our assumption on the proposed structure.We planned two-steps synthesis which included a substitution reaction between 2-(2-bromoethyl)-1,3-dioxalane (an acetal-protected bromopropione aldehyde) and the LTG, followed by a deprotected step of the acetal, utilizing acidic conditions.Comparison between the fragmentation patterns of the synthesized predicted compound (Figure S5E) and the suspected structure of M312 observed in the plants extract sample (Figure S5D) revealed identical mass spectral fingerprint and retention time.The ESI-MS/MS spectra of both M312 and the synthesized predicted compound at a collision energy of 40 eV are displayed in Figure S5D  Although this molecular modification was reported in our previous work by white-rot fungus 4 , its molecular structure is elucidated herein for the first time.Careful interpretation of the mass spectral information observed from the ESI-MS/MS spectra, revealed several product ions representative of the LTG skeleton (fragment ions that observed either in the ESI-MS/MS spectra of LTG or in its other reported conjugates). 1 This support strengthens our assumption that this metabolite is an LTG conjugate.A high intensity product ion at m/z 281.9942, was observed at a collision energy of 40 eV.This product ion was attributed to the loss of a methanol from m/z 314.0206 precursor ion.Since we assumed that one group is a methoxy group (OCH3), the other group should be CO (a complementary to LTG+C2H2O2).Carbamate groups are most likely to dissociate at the C-O bond with the charge remaining on the carbonyl group. 3Protonation occurs at the ether oxygen of the carbamate group, followed by neutral loss of methanol.Therefore, our predicted structure of M314 was the conjugation of a methoxycarbonyl group to one of the three amine nitrogen groups of LTG (Figure S6C).The identification confidence levels of metabolites, based on exact mass measurement and MS/MS fragmentation data as aforementioned for M314, is only at level 3 as documented by Schymansky et al. 5 Since there are still only limited MS/MS spectra in the public databases and the elucidation of M314 structure has not been published in literature.Moreover, a commercially available standard of M314 is not exist, therefore, a home-made synthesis was executed.For the synthesis of M314, methyl chloroformate was selected as a reagent to react with LTG.The detailed synthetic route is provided in the experimental section (Scheme S5).LC-HRMS analysis of peaks generated under reaction conditions, revealed the formation of three main products, two were probably attributed to the mono-substituted LTG and one was attributed to the di-substituted LTG.The formation of the two mono-substituted isomers products was observed due to the presence of more than one nucleophilic site in the molecule.We compared the fragmentation patterns of the two mono-substituted isomeric peaks which observed in the reaction mixture (Figure S6E), to the fragmentation pattern of the peak in the plants extract.
The ESI-MS/MS spectra of the precursor ion at m/z 314.020 of both mono-substituted isomers contained an informative product ion at 281 (loss of methanol), as expected, however, the mass spectral fingerprint of only one isomer was completely identical (masses and their relative abundances, Figure S6E).In addition, the reaction mixture, which contained the two isomeric compounds, was spiked into a control plants extract and the retention times found to be identical.
Ought to the retention time and MS/MS spectra match, we proved that the structure of the M314 in the plant extract and the synthesis candidate is identical and therefore, the structure of M314 has been confirmed with a higher level of confidence.Notably, further investigation is underway to determine the exact position of the substituted nitrogen.Therefore, the identification level is 3.

M418
Mass spectrum of the M418 exhibited a quasi-molecular ion at m/z 418.0675 [M+H] + in positive-ion mode and the molecular formula was identified as C15H18O5N5Cl2 (LTG+ C6H10O5, Table 1) by high resolution MS.The ESI-MS/MS spectra at collision energies (10-50 eV) revealed a single dominant product ion at m/z 256.0148 representative of the intact LTG (data not shown).Full-MS chromatogram along with the ESI-MS/MS spectrum of M418 at a collision energy of 10 eV are depicted in Figures S7A and S7D, respectively.The only information that can be deduced from the ESI-MS/MS spectrum is the difference in mass between the precursor ion at m/z 418.0676 and the product ion at m/z 256.0148.According to literature, N-glucuronidation of LTG pathway is the major route of metabolism in humans as N2-glucuronide and N5-glucuronide. 1 Although N-glucuronidation metabolite m/z 432 was not detected in the plant's extract, a quasi-molecular ion at m/z 418.0675 which might be suitable to the conjugation of glucose to the LTG skeleton was detected.Therefore, our predicted structure of M418 was LTG-N-glucosidation (Figure S7C).Notably, inspection of the LTG structure reveals three possible amino-imino tautomers.The fact that The N2-position is favorable as N2-methyl, N2-oxide and N2-glucuronide metabolites in human may indicate that the basicity the N2-position is high.Therefore, we proposed the LTG-N2-glucosidation structure.

Figure S1 .M271
Figure S1.Identification of LTG-N2-methyl.Full-MS chromatograms of the metabolite in plants extract, r.t = 12.0 min (A) and a commercial standard spiked into a control plants extract (B).A molecular structure (C) the ESI-MS/MS spectrum of LTG-N2-methyl, in plants extract (D) and the ESI-MS/MS spectrum of the commercial standard (E), both at a collision energy of 38 eV.

Figure
FigureS2E, respectively.The confidence level is 3.

Figure S2 .
Figure S2.Identification of M271.Full-MS chromatograms of M271 in plants extract, r.t = 27.4 min (A) and spiked synthesized standard to a control plants extract (B).A suggested molecular structure (C), the ESI-MS/MS spectrum of M271, in plants extract (D) and the ESI-MS/MS spectrum of the synthesized proposed structure of M271 (E), both at a collision energy of 30 eV.

AFigure S3 .M284
Figure S3.Identification of LTG-N2-oxide.Full-MS chromatograms of this metabolite in plants extract, r.t = 14.6 min (A) and spiked synthesized standard to a control plants extract (B).A suggested molecular structure (C), the ESI-MS/MS spectrum in plants extract (D) and the ESI-MS/MS spectrum of the commercial standard of LTG-N2-oxide (E), both at a collision energy of 30 eV.

Figure S4 .M312
Figure S4.Identification of M284.Full-MS chromatograms of M284 in plants extract, r.t = 16.8 min (A) and spiked synthesized standard to a control plants extract (B).The suggested molecular structure (C), the ESI-MS/MS spectrum of M284, in plants extract (D) and the ESI-MS/MS spectrum of the synthesized proposed structure of M284 (E), both at a collision energy of 40 eV.

Figure S5 .M314
Figure S5.Identification of M312.Full-MS chromatograms of M312 in plants extract, r.t = 14.5 min (A) and spiked synthesized standard to a control plants extract (B).The suggested molecular structure (C), the ESI-MS/MS spectrum of M312, in plants extract (D) and the ESI-MS/MS spectrum of the synthesized proposed structure of M312 (E), both at a collision energy of 40 eV.

Figure S6 .
Figure S6.Identification of M314.Full-MS chromatograms of M314 in plants extract, r.t = 21.0 min (A) and spiked synthesized standard to a control plants extract (B).The suggested molecular structure (C), the ESI-MS/MS spectrum of M314, in plants extract (D) and the ESI-MS/MS spectrum of the synthesized proposed structure of M314 (E), both at a collision energy of 40 eV.

Figure S7B )Figure S7 .Figure S10 .M368Figure S11 .M370Figure S12 .M372Figure S13 .M409Figure S14 .M430Figure S15 .M468Figure S16 .
FigureS7B) and the fragmentation patterns (FigureS7Dversus S7E) were compared.The retention time and the mass spectral fingerprint of M418 and the synthesized standard were matched.Although the mass spectral match was based on the presence of a precursor ion at m/z 418.0675 and a single product ion at m/z 256.0148 with a relative intensity of 1~1, it is noteworthy that at collision energies above 50 eV, indicative product ions of the LTG-core were also observed (data not shown).

Figure S31 .
Figure S31.LTG-metabolites that detected in xylem sap during the exposure period.

Table S1 .
Settings for Compound Discoverer 3.0 nodes used in this study.